Check out the NIH Tetramer core facility. Although primarily set up for CD8 T cells, there are a few CD4 types available and they can be custom synthesized. You may be limited in terms of mouse genetic background as well. http://tetramer.yerkes.emory.edu/

The NIH tetramer core facility in the US can make and provide ways to track antigen-specific T cell clones. Most of the ones available are for CD8 T cell clones rather than CD4, but a few were available. It might be worth checking into. This may also be limited by your species of interest and the genetic background of your (mice).

http://tetramer.yerkes.emory.edu/

Thank you for your answer!

I knew this possibility, but even if MHC I tetramers are largely used i know that MHC II tetramers has limitations. Have you direct experience with MHC II tetramers?

A transgenic system with an anti-idiotype antibody is likely to be the most specific means to monitor antigen reactive CD4 T cells and several such systems have been described. If you have a novel system, you may consider developing an ELISPOT approach if you want to quantitate the frequency of reactive cells and something about the nature of their response. You potentially could also expose T cells to antigen-loaded antigen presenting cells ex vivo and identify responder cells based on CFSE dilution. Other methods may also apply. Good luck.

dear Gennarro,

The question you pose is not clear. Do you want to track the cells in vivo or dyou want to measure the immune response of the complete pool of antigen-specific CD4+ T cells. Is it in a setting of immunized mice or in humans? Please be more specific as different answers may apply.

Best regards,

Sjoerd van der Burg

It depends on your system, but one way would be to use teramer technology, which would allow you to identify T cells with receptors specific for your antigen.

Use te tramer specific for the CD4+ population staining versus. CFSE staining in flow cytometry

You mean track by following its destination in vivo? If that is the question and the adoptive transfer of labeled cells is not a possibility, I am afraid you will have to use CD4 Tagged mice, like CD4- GFP, CD4-YFP or CD4 - luciferase mice. However, if you are interested in antigen specific cells, you will have to associate these mice with the antigen - pentamer/ tetramer method EX VIVO, which is analysed by flow cytometry. Well, that`s what comes to my mind....good luck.

Can you use TCR transgenic mice? For example, OTII mouse TCR can recognize only OVA. Thus, if you transfer OTII CD4 T cells into WT B6, you can find OVA antigen specific CD4 T cells after immunization.

This technique of tetramer staining work also without adoptive transfert. I suggest you take out the lymph node or organs that you think you will have the response, then you stain them with CD4+, Tetramer for the challenged epitope (if available) and an activation marker such as CD69 or CD44

You can stain for tetramers loaded with epitope that will be recognized by specific CD4 T cells

Tetramers - see reports from Marc Jenkins

can you describe what you want to accomplish a bit more thoroughly? what kind of system are you working with? In some mouse strains you could use congenic markers (such as THY1.2 and 1.22 or Ly5.1 and 5.2) otherwise labeling cells with CFSE or other cell tracker dyes has been extensively used.

What do you want to look at? Proliferation? Then use CFSE. If not or you want to follow them for a longtime use a dye that doesn't dilute.

The best would be deep sequencing, if you have access to this tecchnology

Specific to what?

There are a few different methods to track the pool of antigen-specific T cells. First, you can simply take spleen or lymph node cells from the immunized mice and stimulate them briefly in vitro with peptide or antigen of interest and then assess their production of cytokine (e.g. IFN-g/TNF-a/IL-2/IL-10) by staining the cells intracellularly with antibodies against the aformentioned cytokines. The trick is that you need to stimulate the cells in the presence of brefeldinA (or monensin in some cases) to keep enough cytokine inside the cells to detect it using flow cytometry. If you use whole antigen - you will need to let the presenting cells in the culture process the antigen (typically overnight) prior to culturing them with the T cells and the brefeldinA (cells can only tolerate brefeldin A for a few hours). Alternatively, you can stain the cells with MHC tetramers if you know the restricting allele and peptide. Making and staining with tetramers to detect CD4+ T cell responses is more challenging than tetramers to detect CD8+ T cell responses, but the NIH tetramer core will make class II tetramers for you if you know the class II allele and the peptide.

An quite easy way to do so is to incubate in vitro whole lymph nod or PBMC stained with CFSE in presence of your antigen for 3/4 days, afterward you make a staining (CFSE)/CD4/CD8/CD3 and Facs them to see the proliferative T cells (CFSElow). This has been done to monitor RSV response in Roux, PlosONE 2008, and I did it also for influenza response. It works very well and allows you to discriminate the CD4 from the CD8 responses. To note : if your antigen is a virus, in vitro restimulation with a live virus will allow you to better observe the reactivation of the T CD8 (better MHC-I presentation) than with inactivated virus.

It depends on the antigen you are using: then you may use cytofluorimetry with tetramers (or directly with the antigen if you have the reagents or you may use functional test with the antigens or block the response to antigens with monoclonals

Thank you to all for the answers!

I'd like to study the CD4 antigen specific immune response in vivo in a mouse model.

To this aim at the moment i'm using adoptive transfer of CFSE labeled OVA-specific transgenic T cells from OT II into recipent mice. I'd like to work in a more physiological condition to study the immune response, cause in a normal mouse i have a few antigen specific clones, after an adoptive transfer i've a very high numbers of it. Does MHC II tetramers works well? I know that MHC I are largely used but MHC II has limitations. At the moment I'm using ovalbumin as model antigen but if i'll find a model that is closest to the physiological immune response i can change it .

I hope that now my question is clear!

Best Regards

Gennaro

I only have direct experience with MHC I tetramers, sorry. Yes, adoptive transfer of CFSE-labelled cells does work well too, but it's very limited for tracking an antigen-specific response. You would have to isolate the cells from a transgenic mouse strain (if available) that ONLY had immune capacity for that particular antigen.

My lab has used MHC class II tetramers extensively and they work very well for many antigens (Tripathi et al J. Immunol. 2007; Wojciechowski et al Eur. J. Immunol. 2006). However, Ova-I-Ab tetramers (for examining OVA-specific responses in BL/6 mice) do not work well at all. They fail to stain the OT-II Tg T cells and therefore likely do not stain a large fraction of the ova-specific population.

Can you provide more information on what exactly you want to do? When you say you want to "track" the CD4 immune response, do you mean you want to find and isolate these antigen-specific T cells or you just want to study how the bulk of T cells (including antigen-specific ones) respond to the antigen? And if so, what do you want to study? Proliferation, cytokine secretion, expression profile, etc.?

All of the systems mentioned above are correct (TCR-transgenic mice, adoptive transfer, T cell serial-dilution, CFSE labeling, ELIspot, microarrays, etc.) but you have to first think of the question instead of how you will answer it.

When you say "track" do you mean you do not want to isolate these cells and measure their function capabilities? You may need in vivo imaging if you want to follow them within the animal itself.

I track endogenous, Ag-specific CD4s in vivo with really good results using the protocol described by Marc Jenkins in Nature Protocols, 2009. Marc and his lab devised a way to enrich a tetramer positive population using MACs sorting and it works very well. OVA is going to be a weak response because the frequency of precursors circulating is really low (in the order of 10-20 cells/mouse) so the expansion is accordingly lower than other epitopes. While the method was described to track small, naive populations (as low as 5-10 cells/mouse), it also serves to track responses really well.

Look up the protocol paper for the details (http://www.ncbi.nlm.nih.gov/pubmed/19373228 and there is also a JoVE video article by Jim Moon, who was a postdoc at Marc's lab). The original article is Moon JJ, Immunity 2007. For examples of how this is used in an infection, see Nelson RW J Immunol 2013 (one of many examples from his lab).

I've attached my own example of a naive and stimulated, endogenous population that is a bit bigger than OVA (the calculated naive frequency shown is 100 cells/naive mouse, but it's more like 50 over N=10).

I forgot to say one thing: there are at least 2-3 registers (2-3 Ag-specific populations) in the OVA-323 epitope. Hence the researcher above that has commented on how the OVA tetramers might not stain all the cells for the response. The NIH now has/will have several tetramers to the different registers. see http://tetramer.yerkes.emory.edu/client/techdocs/I-Ab%20OVA%20242%20packing%20slip.pdf